人脑源性神经营养因子成熟肽基因克隆及表达  

Molecular Cloning and Expression of Human BDNF Gene Encoding Mature Peptide

作　　者：张虎[1] 杨吉成[1] 盛伟华[1] 缪竞诚[1] 李丽娥[1] 王金志[1] 白艳艳[1] 谢宇锋[1] 

机构地区：[1]苏州大学医学院细胞与分子生物学教研室,江苏苏州215007

出　　处：《苏州大学学报（医学版）》2004年第3期326-328,共3页Suzhou University Journal of Medical Science

摘　　要：目的　构建表达人脑源性神经营养因子hBDNF的基因工程菌。方法　人工合成 1对含EcoRI和BamHI限制性酶切位点的特异引物 ,以人血白细胞基因组DNA为模板。应用PCR技术扩增hBDNF成熟肽基因 ,用A T克隆法与 pUCm T载体连接后 ,再定向插入原核表达载体 pBV2 2 0 ,将重组体pBV2 2 0 hBDNF转化大肠杆菌DH5a,经 4 2℃热诱导表达。结果　经限制性酶切和DNA测序证明重组入载体中的DNA片段确为hBDNF ,并成功表达出hBDNF蛋白。结论　该研究成功地克隆出hBDNF基因编码序列并在大肠杆菌中获得稳定表达 ,表达效率达 2 5 % ,为今后生物学活性的研究和神经系统疾病的基因治疗奠定了基础。Objective To construct the genetically engineered bacterium for human brain-derived neurotrophic factor(BDNF). Methods A pair of specific primers including the cleavage site of restriction endonuclease EcoRI and BamHI have been synthesized. By PCR the BDNF gene sequence was directly amplified from the genomic DNA isolated from the human leucocytes. The fragment of resulting gene was then cloned into pUCm-T vector. The insert fragment from the recombinant pUCm-T-hBDNF was directionally ligated with linearied pBV220. DH5a was transformed with the expression recombinant pBV220-hBDNF and induced by heat at 42℃. Results The cloned gene was identified as the sequence encoding hBDNF by restriction anaylsis and DNA sequencing. SDS-PAGE revealed the cloned gene expressed successfully. Conclusion The clone containing the sequence encoding human BDNF is obtained and expressed in DH5a.The highest rate of expression is 25%. This study provides an approach for the study of activity and the gene treatment for the nervous system diseases.

关 键 词：脑源性神经营养因子 PCR A-T克隆 基因表达 

分 类 号：R394.8[医药卫生—医学遗传学] Q782[医药卫生—基础医学]